TNF-driven intracellular complement factor H-NF-κB feed-forward loop amplifies inflammatory mediator production in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) involves multiple pro-inflammatory cytokines; fibroblast-like synoviocytes (FLS) are central effector cells that integrate these signals to produce pathogenic mediators. Tumor necrosis factor (TNF) is a major upstream driver of coordinated inflammatory mediator induction in FLS; however, the underlying mechanisms remain incompletely defined, particularly intracellular feed-forward amplification sustaining and enhancing TNF-driven transcriptional outputs. Complement factor H (FH; gene CFH), a Sushi domain-only complement regulator, is elevated in RA serum. FH is regarded as an extracellular inhibitor of complement activation; however, its potential intracellular expression and functions in FLS have not been explored. We aimed to investigate intracellular FH expression and function using an in vitro transcribed messenger RNA (IVT mRNA)-based approach.

FH expression in sera, synovial tissues, and fibroblast-like synoviocytes (FLS) were assessed by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, RT-qPCR, and immunoblotting. To examine intracellular FH function, FLS were transfected with CFH-encoding IVT mRNA to express FH intracellularly, followed by next-generation sequencing (RNA-seq), RT-qPCR, ELISA, phosphorylation assays, and pathway inhibition. Interactions between TNF-driven signaling and intracellular FH were evaluated using TNF- and/or FH-neutralizing antibodies, with or without CFH-targeting small interfering RNA (siRNA).

FH was highly expressed in RA synovium and predominantly localized intracellularly in FLS. To assess intracellular FH function, FLS were transfected with CFH-encoding IVT mRNA, which induced the coordinated upregulation of RA-relevant mediators, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, IL-6, and multiple chemokines. Mechanistically, intracellular FH activated nuclear factor-κB (NF-κB) signaling, promoting receptor-interacting serine/threonine-protein kinase 2 (RIPK2) phosphorylation and p65 nuclear translocation, and the competitive nucleotide-binding oligomerization domain 2 (NOD2) antagonist suppressed FH-driven gene induction. TNF and intracellular FH additively enhanced pathogenic mediator production. CFH-targeting siRNA attenuated TNF-induced cytokine and chemokine expression; blockade of extracellular FH had no detectable effect. Combined TNF neutralization and CFH knockdown produced more effective suppression of cytokine induction than either intervention alone.

Intracellular FH is a previously unrecognized amplifier of TNF-driven inflammation in RA-FLS via a NOD2/RIPK2/NF-κB axis, establishing a feed-forward loop boosting pathogenic mediator production. Dual targeting of TNF and intracellular FH may represent a therapeutic strategy to suppress synovial inflammation more effectively.